National Food Safety Standard of the People’s Republic of China
GB4789.35-2010Issued by Ministry of Health of the People’s Republic of ChinaGB 4789.35-2010
The Standard substitutes the GB/T 4789.35-2003 “Microbiological Examination for Food Hygiene :Examination of Lactic Acid Bacteria in Beverages Containing Lactic Acid Bacteria”.Compared with the GB/T4789.35-2008, main changes are fol owing:- The count methods of lactic acid bacteria, lactobacil us, bifidobacteria and streptococcusthermophilus are revised.The appendix A of the standard is the normative appendix.The replaced former editions are:- GB 4789.35-1996, GB/T 4789.35-2003, GB/T4789.35-2008
GB 4789.35-2010
Food microbiological examination: Lactic Acid Bacteria
The Standard defines the examination method of lactic acid bacteria in foods containinglactic acid bacteria.The Standard is applicable to the examination of lactic acid bacteria in foods containing livelactic acid bacteria.
The documents referred in the standard are requisite for the application of the standard.For dated references, only the version dated is applicable to the standard.For undated references, the latest version including all modification notices are applicable tothe standard.
3. Terms and Definitions3.1 Lactic acid bacteria
The common name of a group of bacteria capable of fermenting saccharides to producelarge amount of lactic acid.The lactic acid bacteria in the standard are mainly Lactobacil us, Bifidobacterium andStreptococcus genus.
In addition to conventional sterilization and cultivation equipment used in microbiologicallaboratory, other equipment and materials needed are as fol ows:
4.1 Thermostatic Cultivator: 36℃±1℃.4.2 Refrigerator: 2℃~5℃4.3 Homogenizer and sterile homogenizing bag, homogenizing cup or sterile pestle.4.4 Balance: accuracy of 0.1g.4.5 Sterile tube: 18mmX180mm, 15mmX100mm4.6 Sterile pipette: 1ml (with 0.01ml of scale), 10ml(with 0.1ml of scale) or micropipettor andtips.4.7 Sterile conical beaker: 500ml, 250ml
5. Culture Mediums and Reagents5.1 MRS (man rogosa sharpe) Culture Medium and improved MRS with Li-Mupirocin CultureMedium : refer to Appendix A.1.5.2 MC (modified chalmers) Culture Medium: refer to Appendix A.2.5.3 0.5% Sucrose Fermentation Tube: refer to Appendix A.3.5.4 0.5% Cel obiose Fermentation Tube: refer to Appendix A.3.5.5 0.5% Maltose Fermentation Tube: refer to Appendix A.3.5.6 0.5% Mannite Fermentation Tube: refer to Appendix A.3.5.7 0.5% Salicin Fermentation Tube: refer to Appendix A.3.GB 4789.35 -2010
5.8 0.5% Sorbitol Fermentation Tube: refer to Appendix A.3.5.9 0.5% Lactose Fermentation Tube: refer to Appendix A.3.5.10 Aesculin Fermentation Tube: refer to Appendix A.4.5.11 Gram’s Staining Solution: refer to Appendix A.5.5.12 Li-Mupirocin, chemically pure.
6. Examination ProceduresFor the examination procedures of lactic acid bacteria, please refer to Fig. 1.
25g sample (or 25ml)+225ml Sterile Normal Saline
Select 2 to 3 proper dilution degrees, and
Pick bifidobacteria or lactobacil us colonies to inoculate on MRS
agar plate or streptococcus thermophilus on MC agar plate
Figure 1. Examination Procedures of Lactic Acid Bacteria
7. Operation Procedures7.1 Sample preparation7.1.1 The whole process of sample preparation shall observe aseptic operation procedures.7.1.2 Freezing samples shall be thawed at 2℃~5℃ for no more than 18h, and it could also becarried out at a temperature lower than 45℃ for no more than 15 min.7.1.3 Solid and semi-solid food: measure 25g sample with aseptic operation into sterile
GB 4789.35-2010
homogenizing cup containing 225ml normal saline, homogenize at 8000-10000r/m for 1-2 min,and then formulate into 1:10 sample solution; or place it into sterile homogenizing bagcontaining 225ml normal saline, beat with slaping type homogenizer for 1-2 min, and thenformulate into 1:10 sample solution.7.1.4 Liquid sample: First, sufficiently shake the liquid samples, absorb 25ml sample solutionwith sterile pipette into a sterile conical beaker containing 225ml normal saline (proper amountof sterile beads are placed into the beaker in advance), and then shake up to formulate into 1:10sample solution.
7.2 Procedures7.2.1 Absorb 1ml 1:10 sample solution with 1ml sterile pipette or micropipettor, and place into asterile test tube containing 9ml normal saline along the wall of the tube (it is noted that the tip ofthe pipette shall not touch the diluent), shake up the test tube or blow it repeatedly withanother piece of sterile pipette to homogeneity, and then formulate into 1:100 sample solution.7.2.2 Get another 1ml sterile pipette or micropipettor tip, fol ow the above operation proceduresto conduct escalating dilutions on the sample solutions by 10 times. For each time of dilution,one 1ml sterile pipette or tip shall be replaced.7.2.3 Lactic acid bacteria count7.2.3.1 Lactic acid bacteria count
According to the estimation of live bacteria count to be tested, select 2 to 3 propercontinuous dilution degrees, for each dilution degree, absorb 0.1ml of dilution sample andplace it onto 2 MRS agar plates, and then spread the surface with L-shape bar. Count alcolonies on the plate after anaerobic incubation at 36’C+/-1’C for 48hr+/-2hr.The whole experiment process shall be completed within 15minutes.
According to the estimation of live bacteria count to be tested, select 2 to 3 propercontinuous dilution degrees, for each dilution degree, absorb 0.1ml of dilution sample andplace it onto 2 updated MRS agar plates with Li-Mupirocin, and then spread the surface withL-shape bar. Count al colonies on the plate after anaerobic incubation at 36’C+/-1’C for48hr+/-2hr.The whole experiment process shall be completed within 15minutes.
According to the estimation of live bacteria count to be tested, select 2 to 3 propercontinuous dilution degrees, for each dilution degree, absorb 0.1ml of dilution sample andplace it onto 2 MC agar plates, and then spread the surface with L-shape bar. Do colonycount after aerobic incubation at 36’C+/-1’C for 48hr+/-2hr. The colony morphology ofstreptococcus thermophilus on the MC agar plate: the colony is medium in size and less thannormal. The colony is round and red, the diameter is 2mm+/-1mm, and the back of thecolony is pink.The whole experiment process shall be completed within 15minutes.
The lactobacil us count equals lactic acid bacteria minus bifidobacteria 7.2.3.2, and minusstrephtococcus thermophilus count 7.2.3.3.
It could be observed with naked eyes, apply magnifying glass or bacteria colony counter
GB 4789.35 -2010
when necessary, and record the dilution times and corresponding plate count. Plate countnumber is represented by colony-forming units (CFU).
7.3.1 Select the plates for the number of colonies between 30CFU and 300CFU, and withoutspreading growth on the plate. For plate with colony count under 30CFU, the number of colony isrecorded, while for plate count over 300, it shall be recorded as uncountable. For each dilutiondegree, the average number of two plates shall be applied.7.3.2 For those plates with large piece of colony growing, they shall not be applied. If the piece ofcolony covers less than one half of the plate area, and the colonies on the remaining half of theplate area scatter evenly, it shal be counted of this half of the plate and then multiply by 2, torepresent the entire plate count.7.3.3 When there occurs chain like growth on the plate without evident border line betweencolonies on the plate, each chain shal be calculated as one colony.
7.4 Formulation of results7.4.1 If there is only one dilution degree whose plate count fall in the proper counting scope, theaverage plate count of both plate shall be calculated, and then multiply the average value bycorresponding dilution times, to serve as the total plate count in one gram (or ml) of sample.7.4.2 If there are two continuous dilution degrees whose plate count falls in the propercounting scope, they shall be calculated as in Formula (1):
- The total number of colonies on the plates (including the plates within the range of
proper plate count;N1 - The number of colonies on the plates of the first proper dilution degree;N2 - The number of colonies on the plates of the second proper dilution degree;d - Dilution Factor (the first dilution degree).
7.4.3 If the colony numbers on the plates of all dilution degrees are all over 300CFU, count theplates with the maximum dilution degree. For other plates, they shall be recorded asuncountable, and the results shall be obtained by multiplying the average colony number by themaximum dilution times.7.4.4 If the colony numbers on the plates of all dilution degree are all less than 30CFU, it shall becalculated by multiplication of average colony number on the minimum dilution degree plates bythe dilution times.7.4.5 If, for plates of all dilution degrees (including the original liquid samples), there is no colonygrowth, then it shall be calculated as multiplying the minimum dilution degree by a factorsmal er than 1.7.4.6 If, for plates of all dilution degrees, the colony number fal s outside the range between30CFU and 300CFU, part of which are less than 30CFU or more than 300CFU, then it shall becalculated for the plates whose colony number is closest to 30CFU or 300CFU, as the averagecolony number multiply by dilution times.
7.5 Colony count report7.5.1 When the plate count falls within 100CFU, it shall be rounded up and reported as integer.GB 4789.35-2010
7.5.2 When the plate count is larger than or equal to 100CFU, the third digit shall be rounded up,and take the first two digits, while the fol owing digits are replaced by 0; it could also beindicated as exponential of 10CFU, round-up and then take the two significant digits.7.5.3 For sampling by weight, CFU/g is applied as the report unit, while for sampling by volume,CFU/ml is applied as the report unit.
8. Results and reportsTo issue the report cccording to the colony count, the unit of report is CGU/g(ml)
9. Lactic acid bacteria identification (Optional)9.1 Pure incubationPick 3 or more separate colonies, streptococcus thermophilus shall be inoculated on MC agarplates, while lactobacil us shall be inoculated on MRS agar plates. Then, incubate underfacultative anaerobic conditions at 36℃±1℃ for 48 hours.9.2 Identification9.2.1 The identification of bifidobacteria shall be subject to GB/T 4789.34.9.2.2 Smear slide microscopy: lactobacil us is multiple in morphology, in elongated shape,bending rod shape or short rod shape. It has no spore, and is positive in Gram’s Staining.Streptococcus thermophilus is in ball shape or clavate shape, with a diameter between 0.5umand 2.0um, distributed in pairs or chain, without spore, and it is positive in Gram’s Staining.9.2.3 For the main biochemical reaction of lactic acid bacteria, pls refer to Table 1 and Table 2.
Table 1. Biochemical reactions within common lactobacilius genus
Aesculin Cel obiose Maltose Mannite Salicin
Note: +indicates over 90% of the strains mentioned above are positive; -indicates over90% of the strains mentioned above are negative; while ND indicates not detectable.
Table 2. The main biochemical reactions in streptococcus thermophilus
Synanthrin Lactose Mannite Salicin Sorbitol Hippuric Aesculin
Note: + indicates over 90% of the strains mentioned above are positive; - indicates over90% of the strains mentioned above are negative.GB 4789.35 -2010
A.1.2 Preparation methodPut the ingredients mentioned above into 1000ml distil ed water, heat for dissolving. Adjust thepH value, autoclave at 121℃ for 15-20min after distribution.A.1.3 updated MRS with Li-MupirocinA.1.3.1 preparation of Li-Mupirocin reserve solution: weigh 50mg Li-Mupirocin into 50ml distil edwater, sterilization by filter of 0.22um micro-hole filterable membrane.A.1.3.2 Preparation methodPut the A.1.1 ingredients into950ml distil ed water, heat for dissoclving. Ajesut the pH value,autoclave at 121℃ for 15-20min after distribution.Cool down to 48℃ in water bath, then use injector with 0.22um micro-hole filterable membraneto inject Li-Mupirocin reserve solution into melting agar. The concentration of Li-Mupirocinshould be 50ug/ml.
A.2.2 Preparation methodPut the former 7 ingredients into distil ed water, heat for dissolving. Adjust the pH value, and
GB 4789.35-2010
then add into the neutral red solution. Autoclave at 121℃ for 15-20min after distribution.
A.3 Lactobacil us Saccharides Fermentation TubeA.3.1 Ingredients
1.6% Bromocresol purple ethanol solution 1.4mlDistil ed Water
A.3.2 Preparation methodAdd saccharides needed per 0.5%, and then distribute into smal test tubes. Autoclave at 121℃for 15-20min.
A.4 Aesculin Fermentation TubeA.4.1 Ingredients
A.4.2 Preparation methodDissolve the ingredients mentioned above into distil ed water, and then autoclave at 121℃ for15-20min.
A.5 Gram’s Staining SolutionA.5.1 Crystal Violet Staining SolutionA.5.1.1 Ingredients
A.5.1.2 Preparation methodDissolve the crystal violet completely into ethanol, and then mix up with ammonium oxalatesolution.A.5.2 Gram’s Iodine SolutionA.5.2.1 Ingredients
A.5.2.2 Preparation methodFirst mix iodine and potassium iodide together, and then add into smal amount of distil ed water,and shake up sufficiently, after complete dissolution, add distil ed water to it to 300ml.A.5.3 Safranin Restaining SolutionA.5.3.1 Ingredients
GB 4789.35 -2010
A.5.3.2 Preparation methodDissolve the safarin into ethanol, and then dilute with distil ed water.
A.5.4 Staining MethodA.5.4.1 Fix the smear slide on the flame of alcohol burner, drip crystal violet staining solution,dye for 1 min, and then rinse with water;A.5.4.2 Drip the Gram’s Iodine Solution，react for 1 min, and then rinse with water;A.5.4.3 Drip 95% of ethanol to decolor for about 15~30s, until the staining solution is washed off,and it shal not be overdone, and then rinse with water;A.5.4.4 Drip the restaining solution, restain for 1min. Rinse with water, and then dry it formicroscopy.

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